The present invention relates to a class of novel analogs of porcine growth hormone. In particular, the present invention pertains to recombinantly-produced analogs of porcine growth hormone wherein one or more residues corresponding to the residues at positions 32 through 38 in the amino acid sequence of naturally occurring porcine growth hormone are deleted. The present invention further relates to compositions containing such analogs and to the use of such analogs and compositions to increase the growth of mammals.
The pituitary gland of normal mammals produces and secretes into the bloodstream a substance called growth hormone ("GH"). The amino acid sequences of human ("hGH"), bovine ("bGH"), and porcine ("pGH") growth hormones are similar. See Dayhoff, Atlas of Protein Sequence and Structure, Volume 5, Supplement 6, National Biomedical Research Foundation, Washington, 120-121 (1976); and Seeburg et al., DNA, 2, 37-45 (1983). The amino acid and nucleotide sequences of salmon growth hormone ("sGH") is also known, Sekine et al., Proc. Nat'l. Acad. Sci. (USA), 82, 4306-4310 (1985). Based upon an alignment of the sequences of bGH, hGH, evine growth hormone, pGH, and sGH which provides the highest degree of homology among these growth hormones, certain highly conserved regions may be identified. See e.g., Dayhoff, supra, and Sekine et al., supra.
In vivo, growth hormone promotes construction of protein from amino acids, an initial fall in plasma glucose upon administration, a gradual rise in plasma glucose after the initial fall, and a breakdown of fats into fatty acids. The effects associated with growth hormone are respectively referred to as growth promotion (i.e., weight gain), insulin-sparing, diabetogenic and lipolytic effects. An antilipolytic effect has also bee-n reported, but this appears to be a facet of the insulin-like activity of the hormone. Goodman, Metabolism, 19, 849-855 (1970).
In addition, growth hormones are similar in structure to lactogenic hormones and are capable of inducing similar effects. For example, hGH differs from the human placental lactogen at about 15% of its residues. Wallis et al., in Growth Hormone and Related Peptides, Pecile et al., eds., Excerpta Medica, Amsterdam, 1-13 (1976). Human growth hormone differs from human prolactin at about 25% of its residues. Wallis et al., supra. Subcutaneous injection of bGH or recombinant bGH ("rbGH") increases milk yield in cows, goats and sheep. Eppaird et al., J.Dairy Sci. 68, 1109-1115 (1985); Bauman et al., J. Dairy Sci., 68, 1352-1362 (1985); Hart, Proc. Nutr. Soc., 42, 181-194 (1983); and see Hart et al., Biochem. J., 218, 573-581 (1984).
The isolation of growth hormone from pituitaries involves lysing pituitary cells associated with production of the hormone. However, the lysing of cells releases proteolytic enzymes (proteases) which may cleave at least some of a naturally-occurring pituitary growth hormone ("nGH") into fragments. Furthermore, once secreted into the bloodstream, nGH is exposed to proteases which may cleave nGH into the same or into different fragments. A major area of investigation for growth hormone fragment research is directed at a determination of whether nGH or its fragments or both give rise to the actions associated with growth hormones which have been extracted or which are circulating in the bloodstream. In this regard, it may be noted analogs of hGH rendered resistant to digestion by the protease trypsin by chemically modifying lysine or arginine residues possess significant, albeit attenuated, growth-promoting, diabetogenic and insulin-like activities. Cameron et al., Biochim. Biophys. Acta, 254-260 (1985). Nevertheless, discrete portions ("domains") of the nGH molecule are believed to be responsible for one or another of the effects of nGH. To the extent that responsibility for the actions of nGH may be localized in this way, fragments and analogs may be produced in which the protein-synthetic, insulin-sparing, diabetogenic and lipolytic effects are selectively altered.
As used hereinafter, the positions of amino acid residues present in fragments or analogs of growth hormone are identified in a subscript wherein numbers indicate the presence of the residues found at the same positions in the corresponding nGH and wherein deletions are indicated by a comma. For example, naturally occurring porcine growth hormone is represented by pGH.sub.1 -190.
A 20,000-dalton variant ("20K") of hGH (22,000-dalton) which may be isolated from pituitaries and which corresponds to hGH.sub.1 -31,47-191 promotes growth in hypophysectomized rats, is not hyperglycemic or hyperinsulinemic in dogs, is neither insulin-sparing nor lipolytic in vivo or in vitro, and is less reactive in radioimmunoassays for hGH than is hGH itself. Lewis et al., J. Biol. Chem., 253, 2679-2687 (1978); Frigeri et al., Biochem. Biophys. Res. Commun., 91, 778-782 (1979); Lewis et al., Biochem. Biophys. Res. Commun., 92, 511-516 (1980); and Lewis et al., Endocr. Res. Commun., 8, 155-164 (1981). The 20K variant of hGH is a product of post-transciptional modification. Lewis et al., Biochem. Biophys. Res. Commun., supra. It may be the case that the 20K variant of hGH may be a more important growth promoter than would be predicted from its in vitro bioactivity due to its tendency to dimerize and thus escape renal degradation. Baumann et al., Endocrinology, 117, 1309-1313 (1985).
Fragments of hGH which include residues deleted from 20K hGH have also been disclosed. Although none of these fragments are reported to promote growth, some exhibit properties of potential relevance to the diabetogenic and lipolytic properties of growth hormone.
A synthetic fragment corresponding to residues 31-44 of hGH is lipolytic in vivo in starved animals and in vitro [Yudaev, et al., Biokhimiya, 41, 843-846 (1976)] but stimulates glucose uptake (i.e. was insulin-sparing) only after in vitro preincubation in the absence of GH, a non-physiological state. Yudaev, et al., Biochem. Biophys. Res. Commun., 110, 866-872 (1983). Some peptides analogs of hGH are diabetogenic but an analog of hGH.sub.52 -77 is not. Lostroh, et al., Diabetes, 27, 597-598 (1978). A peptide consisting of hGH.sub.20 -41 is devoid of activity. Reagan, Diabetes, 27, 883-888 (1978). A peptide consisting of hGH.sub.1 -36 is devoid of effect on blood glucose or on growth. Chillemi, et al., in Growth Hormone and Related Peptides, Pecile, et al., eds., Excerpta Medica, Amsterdam, 50-63, (1976).
However, a peptide corresponding to hGH.sub.32 -46 causes a decrease in serum free fatty acids, and is insulin-sparing when coadministered with insulin in vitro [Frigeri et al., in Proceedings, 64th Annual Meeting of the Endocrine Society, San Francisco, 101 (Abstract 88) (1982)] and in vivo [Rudman, U.S. Pat. No. 4,558,033, and Stevenson et al., Diabetes, 33, 149A (Abstract No. 572) (1984)]. Fragments and analogs (involving substitution of heterologous amino acids or stereoisomers) of hGH.sub.32 -46 are also insulin-sparing when coadministered with insulin in vivo. Jones et al., copending and coassigned U.S. patent application Ser. No. 501,024 now U.S. Pat. No. 4,699,897.